Myocardial grafts and cellular compositions useful for same

ABSTRACT

Described are preferred myocardial grafts of skeletal myoblasts or cardiomyocytes, and cellular compositions and methods useful in obtaining the grafts. The myocardial grafts are stable and can be used, for example, to deliver recombinant proteins directly to the heart.

BACKGROUND OF THE INVENTION

[0001] The present invention resides generally in the field ofcardiology, and more particularly relates to stable myocardial graftsand methods and cellular compositions useful for achieving such grafts.

[0002] As further background, organ transplantation has been widely usedto replace diseased, nonfunctional tissue. More recently, cellulartransplantation to augment deficiencies in host tissue function hasemerged as a potential therapeutic paradigm. One example of thisapproach is the well publicized use of fetal tissue in individuals withParkinsonism (reviewed in (1), see reference list, infra.), wheredopamine secretion from transplanted cells alleviates the deficiency inpatients. In other studies, transplanted myoblasts from uneffectedsiblings fused with endogenous myotubes in Duchenne's patients;importantly the grafted myotubes expressed wild-type dystrophin (2).

[0003] Despite their relevance in other areas, these earlier studies donot describe any cellular transplantation technology which can besuccesfully aplied to the heart, where the ability to replace damagedmyocardium would have, obvious clinical relevance. Additionally, the useof intra-cardiac grafts to target the long-term expression of angiogenicfactors and ionotropic peptides would be of therapeutic value forindividuals with myocardial ischemia or congestive heart failure,respectively.

[0004] In light of this background there is a need for the developmentof cellular transplantation technology in the heart. Desirably, suchtechnology would not only provide stable grafts in the heart but alsoenable the delivery of useful recombinant proteins or other moleculesdirectly to the heart. The present invention addresses these needs.

SUMMARY OF THE INVENTION

[0005] The applicant has established cellular grafts in the myocardiumwhich are viable long-term. Cardiomyocytes and skeletal myoblasts havebeen grafted directly into the myocardium of syngeneic animals. Viablegrafts were detected at least one-half year post-implantation (thelatest time point assayed). The presence of the grafts was notaccompanied by overt cardiac arrhythmia, and the majority of the graftswere juxtaposed directly to the host myocardium and not encapsulated. Ithas thus been discovered that the myocardium can serve as a stableplatform for cellular transplants. These transplants can be used for thelocal delivery of recombinant molecules to the heart and/or forreplacing diseased tissue to supplement myocardial function.

[0006] Accordingly, one preferred embodiment of the invention provides amyocardial graft in an animal which includes a stable graft of skeletalmyoblasts or cardiomyocytes incorporated in myocardial tissue of theanimal.

[0007] Another preferred embodiment of the invention provides a methodfor forming a stable myocardial graft in an animal. The inventive methodincludes the step of introducing skeletal myoblasts or cardiomyocytes inmyocardial tissue of the animal so as to form a stable myocardial graft.The cells can be conveniently introduced, for example, by injection.

[0008] Another preferred embodiment of the invention provides a methodfor delivering a recombinant molecule to myocardial tissue of an animal.This method includes the step of establishing a stable graft of skeletalmyoblasts or cardiomyocytes incorporated in myocardial tissue of theanimal, wherein the myoblasts or cardiomyocytes deliver the recombinantmolecule to the myocardial tissue. In this embodiment the myoblasts orcardiomyocytes will carry transgenes encoding the recombinant molecule.

[0009] Another preferred embodiment of the invention provides a cellularcomposition comprising a substantially homogeneous population ofnon-immortalized cardiomyocytes. This and other cell populations can beobtained utilizing a preferred inventive method that includes (i)transfecting embryonic stem cells to introduce a marker gene enablingselection of one cell lineage from other cell lineages resulting fromdifferentiation of the stem cells, (iii) causing the stem cells todifferentiate, and (iv) selecting said one cell lineage based on themarker gene. The cells used in and resulting from such methods also forma part of the present invention.

[0010] Still another preferred embodiment of the invention provides anon-human animal having a stable graft of skeletal myoblasts orcardiomyocytes incorporated in myocardial tissue of the animal.

[0011] The invention thus provides myocardial grafts, methods andcellular compositions useful for forming myocardial grafts, and animalswhich have the myocardial grafts. The grafts will find use both as avehicle for delivering therapeutic substances such as recombinantproteins and other molecules, and as a means for replacing diseasedtissue to supplement myocardial function. Cellular compositions of theinvention can be used directly to prepare grafts, and will also beuseful in screening drug substance effects on cardiomyocytes and forexpressing and obtaining recombinant proteins. Grafted animals can beused, for example, to screen the effects of recombinant molecules on theheart.

[0012] These and other objects and advantages of the invention will beapparent from the following description.

BRIEF DESCRIPTION OF THE FIGURE

[0013]FIG. 1 is a schematic diagram illustrating DNA used to generateMHC-nLAC transgenic mice in Example 3, infra.

DESCRIPTION OF THE PREFERRED EMBODIMENT

[0014] For the purpose of promoting an understanding of the principlesof the invention, reference will now be made to certain embodimentsthereof and specific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of theinvention is thereby intended, such alterations, further modificationsand applications of the principles of the invention as illustratedherein being contemplated as would normally occur to one skilled in theart to which the invention relates.

[0015] As indicated above, the present invention provides stablemyocardial grafts of skeletal myoblasts and/or cardiomyocytes. In thisregard, as used herein the term “stable myocardial graft” is intended tomean a myocardial graft whose cells are viable for a period of at leastabout 2 weeks. Surprisingly, such stable grafts have been readilyachieved in accordance with the invention, with preferred grafts havingcells viable for six months or more. Myocardial grafts of the inventioncan thus provide for long-term delivery of recombinant proteins or othermolecules to the heart and/or long-term supplementation of myocardialtissue.

[0016] The skeletal myoblasts and cardiomyocytes used in the inventioncan be obtained or isolated from any suitable source. Skeletalmyoblasts, including for example C2C12 skeletal myoblasts, are availablefrom public depositories such as the American Type Culture Collection(ATCC) (Rockville, Md.). Skeletal myoblasts can also be isolated fromskeletal muscle using techniques well known to the art and literature.Cardiomyocytes useful for the invention can be obtained using techniquesdescribed in the literature (3) or using methods described moreparticularly in the Examples below. Briefly, one such method involvesdigestion of heart tissues to obtain cardiomyoctes.

[0017] Another method involves using an appropriate marker to selectspecific cell lineages, such as cardiomyocytes, from other cell lineagesresulting from the differentiation of embryonic stem cells (totipotentcell lines derived from the inner cell mass of blastocysts as describedin (22)). The preferred method involves a positive selection scheme.Thus, a marker gene, such as a gene conferring antibiotic resistance(e.g. neomycin or hygromycin), is introduced into the stem cells underappropriate control such that expression of the gene occurs only in thedesired cell lineage. For example, the marker gene can be under thecontrol of a promoter which is active only in the desired cell lineage.Upon differentiation of the stem cells, the desired lineage is thenselected based upon the marker, e.g. by contacting the mixeddifferentiated cells with the appropriate antibiotic to which thedesired lineage has been conferred resistance. Cell lines other than thedesired line will thus be killed, and a substantially pure, homogeneouspopulation of the desired line can be recovered. In more preferredmethods, two markers are introduced into the parent stern cells, oneallowing selection of transfected stem cells from non-transfected cells,and one allowing selection of the desired cell lineage from otherlineages. A double positive selection scheme can thus be used where eachselectable marker confers antibiotic resistance. Using this selectionmethodology, populations comprised about 90% and even about 95-100% ofthe desired cell lineage can be obtained, as demonstrated in theExamples below.

[0018] To obtain grafts of the invention, the skeletal myoblasts orcardiomyocytes will be introduced into the myocardial tissue of a livinganimal such as a mammal. The cells can be introduced in any suitablemanner, but it is preferred that the mode of introduction be asnon-invasive as possible. Thus, delivery of the cells by injection,catheterization or similar means will be more desired.

[0019] The resulting graft-bearing animals have exhibited normal sinusrhythms, indicating that the graft per se, as well as the graft-hostmyocardium border zone, does not induce arrhythmias. This is in starkcontrast to the remodeling that frequently occurs following infarcts inhumans; the border zone of the infarct may give rise to circus loopswhich result in clinically significant arrhythmias (4, 5).

[0020] Grafts of the invention can be proliferative ornon-proliferative. For example, the AT-1 grafts established in thespecific Examples below are proliferative. On the other hand, theskeletal myoblast-derived grafts formed in the Examples arenon-proliferative, with the absence of tritiated thymidine uptakedemonstrating that the formation of stable intra-cardiac grafts was notdependent upon sustained cell proliferation.

[0021] Preferred grafts will be characterized by the presence of directintracellular coupling and the formation of gap junctions between hostand grafted cells. Moreover, such grafts will not cause immune responsein the host, and will exhibit terminal differentiation of grafted cellsand a non-tumorogenic nature.

[0022] Grafts of the invention are useful inter alia to delivertherapeutic proteins and the like via secretion from grafted cells, andto replace diseased or damaged tissue to supplement myocardial function.As examples of therapeutic protein deliveries, grafts may expressangiogenic factors (as exemplified by basic and acidic Fibroblast GrowthFactor; Transforming Growth Factor-Beta, Vascular Endothelial GrowthFactor and Hepatocyte Growth Factor) to induce neovascularization.Similarly, grafts expressing neurotrophic agents near an infarctedregion may be used to ameliorate the arrhythmogenesis associated withthe border zone. These and many other candidate substances for targeteddelivery to the heart will be apparent to those skilled in the area.

[0023] To promote a further understanding of the invention and itsprinciples and advantages, the following specific Examples are provided.It will be understood that these Examples are illustrative, and notlimiting, in nature.

EXAMPLE 1 Generation of Stable AT-1 Cardiomyocyte Grafts

[0024] A. Methods

[0025] AT-1 Cell Culture and Myocardial Grafting Protocol.

[0026] AT-1 cardiomyocytes were isolated from subcutaneous tumors bysequential collagenase digestion and cultured in PC-1 medium (Ventrex,Coon Rapids Minn.) containing 10% fetal calf serum-as previouslydescribed in (6). Cells were labeled with 10 μM8-chloromethyl-4,4-difluoro-1,3,5,7,-tetramethyl-4-bora-3a,4a-diazaindecene(BODIPY, Molecular Probes, Eugene Oreg.) for 30 min at 37° C. tofacilitate localization of the injection site. Immediately beforeinjection, cells were harvested with trypsin and collagenase, washedthree times with serum-free PC-1 medium and directly injected into theventricular myocardiurn of syngeneic B6D2/F1 mice (Jackson Laboratories,Bar Harbor Mass.) under open heart surgery as described in (7). Cells(4-10×10⁴) were injected in a volume of 2-3 μl using a plastic syringefitted with a 30 gauge needle.

[0027] Histology

[0028] Hearts were removed following cervical dislocation andcryoprotected in 30% sucrose, embedded and sectioned at 10 μm with acryomicrotome as described (8). For hematoxylin and eosin (H and E)staining, sections were post fixed in acetone:methanol (1:1) and stainedaccording to manufacturer's specifications (Sigma Diagnostics, St. LouisMo.). For immuno-histology, unfixed sections were reacted withpolyclonal rabbit anti-T-Ag antibodies (either 161-T, see (3) or 162-T)followed by horseradish peroxidase-conjugated goat anti-rabbit antisera(Boehringer Mannheim, Indianapolis Ind.), and visualized bydiaminobenzidine reaction with nickel enhancement as described in (9).Monoclonal antibodies against the common leukocyte antigen (CD45;antibody M1/9.3HL, Boehringer Mannheim) and against the macrophage Mac-1antigen (CD11b; antibody M1/70HL, Boehringer Mannheim) were used tomonitor intra-cardiac graft rejection. The Mac-1 antibody has 75-90%cross reactivity with lymphocytes. After treatment with primaryantibody, sections were incubated with horseradish peroxidase-conjugatedrabbit anti-rat antisera (Boehringer Mannheim), and visualized bydiaminobenzidine reaction with nickel enhancement. For [³H]-thymidineincorporation, mice were given a single bolus injection of isotope (400μCi at 28 Ci/mM, Amersham, Arlington Heights Ill.) and eighteen hourslater sacrificed by cervical dislocation. The heart was removed,cryoprotected in 30% sucrose, embedded and sectioned with acryomicrotome. Sections were post-fixed in methanol:acetone (1:1),stained with H and E, and a thin layer of photographic emulsion (IlfordL.4, Polysciences, Warrington Pa.) diluted 1:1 with distilled water wasapplied. Sections were exposed for 5-7 days at 4° C., and developed inKodak D-19 at 20° C. for 4 minutes, washed with distilled water for 1minute, fixed in 30% sodium thiosulfate for 10 minutes, and washed indistilled water.

[0029] Electron Microscopy (EM).

[0030] Tissue blocks were fixed in 2% glutaraldehyde in 0.1M cacodylatebuffer (pH 7.4) and post-fixed in 2% osmiuin tetroxide (StevensMetallurgical Corp., New York N.Y.). All other EM chemicals wereobtained from Ladd Research Industries, Inc. (Burlington Vt.). Tissuewas stained en bloc with 2% uranyl acetate in pH 5.2 maleate buffer(0.05 M), dehydrated, and embedded in Ladd LX-112. Grafts were locatedusing 1 μm sections stained with toluidine blue. After trimming, theblock was thin sectioned, and stained with uranyl acetate and leadcitrate. Specimens were viewed on a Phillips 400 transmission electronmicroscope.

[0031] Electrocardiogram (ECG) Analyses.

[0032] For surface ECG records, mice were anesthetized (2.5% Avertin,0.015 ml/g body weight, IP, Fluka Chemicals, Lake Ronkomkoma N.Y.),surface electrodes were placed in the standard lead 1 position, and ECGswere recorded with a Narco Biosystems (Houston Tex.) high gain amplifiercoupled to an A/D converter (Coulbourn Instruments, Lehigh Valley Pa.).

[0033] Plasma Enzyme Assay (PEA).

[0034] For lactate dehydrogenase (LDH) isoform assay, plasma wasisolated by retro-orbital sinus bleeds under anesthesia (2.5% Avertin,0.015 ml/g body weight, intraperitoneally (IP)). Plasma was fractionatedon 1% agarose gels (CK Isoenzyme electrophoresis system, CIBA-CorningDiagnostics, Corning N.Y.) and the LDH isoforms visualized by aTNBT-Formazan histochemical assay (LDH Assay Kit, Sigma Diagnostics, St.Louis Mo.).

[0035] B. Results

[0036] In these studies, AT-1 cardiomyocytes (derived from transgenicanimals that expressed the T-Ag oncoprotein in the heart) were injecteddirectly into the myocardium of syngeneic mice and the viability of thegrafted material was assessed. To facilitate localization of theinjection site in preliminary experiments, AT-1 cardiocytes wereincubated briefly with BODIPY prior to grafting. BODIPY is a nontoxicglutathione reactive dye which permits fluorescent tracking of livingcells. The graft site was easily visualized by fluorescence microscopyusing a FITC cube. Subsequent experiments did not utilize BODIPY.

[0037] Fifty percent (14/28) of the animals receiving AT-1 cardiomyocyteinjections developed intra-cardiac grafts. In most instances, the graftswere neither encapsulated nor surrounded by scarred myocardium. At thelevel of light microscopy, grafted AT-1 cardiomyocytes were observeddirectly juxtaposed with host cardiomyocytes. The identity of the AT-1cardiomyocytes was confirmed by immuno-peroxidase assay using ananti-T-Ag antibody primary antibody (162-T) followed by a horseradishperoxidase conjugated secondary antibody. Specificity of the anti-T-Agantibody has been established previously (10, 11). Black precipitate towas observed over cardiomyocyte nuclei in the graft but not in the hostmyocardium, confirming that the graft was comprised of AT-1cardiomyocytes. Similar results were obtained with other anti-T-Agantibodies, and no signal was observed in the absence of primaryantibody.

[0038] Viable AT-1 cardiomyocytes were observed at least as long as fourmonths post-implantation. During this period, some degree of graftproliferation occurred; ³[H]-thymidine incorporation analyses detectedDNA synthesis in the grafted cells. Ten percent of the AT-1cardiomyocyte nuclei were synthesizing DNA as evidenced by isotopeincorporation into the nucleus. However, the rate was appreciably lessthan that observed for cultured AT-1 cardiomyocytes, where 50% of thecells synthesized DNA following a similar ³[H]-thymidine pulse. Inseveral instances, the grafted AT-1 cardiomyocytes were localized withinthe subpericardial space.

[0039] Immunohistologic experiments were employed to determine if theintra-cardiac grafts were subject to chronic rejection. Grafts olderthan one month failed to react with antibodies specific for mouseleukocytes; signals observed in blood vessels located on the samesection provided a positive control for the experiment. Similarly, anantibody which detects mouse macrophages and lymphocytes did not reactwith the intra-cardiac graft; once again positive signal was observed ina blood vessel located on the same section. Collectively, these resultsindicate the absence of chronic graft rejection by the syngeneic hosts.This result is supported by the observation that cyclosporine treatment(50 mg/kg body weight, administered intraperitoneally daily) did notinfluence significantly the frequency of intra-cardiac grafting (50%success rate, n=6). Sex of the host animal also did not appear toinfluence significantly the rate of graft formation (46% success rate inmales, n=13; 53% success rate in females, n=15). The frequency ofgrafting was similar in animals examined at early time points (1-40 dayspost-grafting, 47%, n=15) as compared to those examined at later timepoints (40-120 days post-grafting, 54%, n=13). Finally, similarfrequencies of intra-cardiac grafting were observed when cells weredelivered to either the left ventricular free wall or the apex of theheart.

[0040] Electron microscopic analysis of the AT-1 cardiomyocyte graftsconfirmed the absence of encapsulation. High power views revealedwell-developed junctional complexes between adjacent cells within thegraft. Graft cardiomyocytes contained numerous polyribosomes and thededifferentiated myofibrillar ultrastructure typical of AT-1 tumors invivo (6). Electron-dense secretory granules were also observed in theAT-1 cardiomyocyte grafts, as would be expected for myocytes of atrialorigin. Host cardiomyocytes bordering the grafts had normalultrastructure with well-formed sarcomeres. Although only a thinbasement membrane separated AT-1 and host cardiomyocytes, no junctionalconnections between these two cell types were observed.

[0041] Surface electrocardiograms were performed to determine if thepresence of AT-1 cardiomyocyte grafts influenced the autonomic rhythm.No appreciable differences were observed between records from shamanimals and those which harbored grafts. In each case, the experimentalanimals exhibited normal sinus rhythm, with an anesthetized heart rateof approximately 400 beats per minute. Normal P-QRS coupling wasmaintained, indicating that the grafted AT-1 cardiomyocytes did not actas an ectopic pacemaker. This latter result is important in light of theobservation that AT-1 cardiomyocytes exhibit spontaneous electricalactivity both in vivo (12) and in culture (3). The absence of overtarrhythmia also indicated that graft-induced myocardial remodeling wasnot associated with the generation of significant circus rhythms.

[0042] In addition to surface ECG, plasma LDH levels were assessed inmice carrying AT-1 cardiomyocyte grafts. The presence of the cardiac LDHisoform in the circulation is a well established hallmark of myocardialinfarction. No cardiac LDH (isoform-1) was apparent in mouse plasmaprior to grafting. After the introduction of AT-1 cardiomyocytes, therewas a transient appearance of the cardiac isoform in the plasma, whichmost likely reflected damage to the host myocardium as well as damagedAT-1 cardiomyocytes. A transient increase in plasma skeletal LDH isoformwas also observed following grafting surgery, presumably reflectingdamage caused by the trans-thoracic incision. The plasma LDH profilesreturned to normal by 7 days post-implantation. Thereafter, the plasmaLDH profiles remained normal despite the presence of grafts.

EXAMPLE 2 Generation of Stable C2C12 Myoblast Grafts

[0043] A. Methods

[0044] C2C12 Cell Culture and Myocardial Grafting Protocol.

[0045] C2C12 myoblasts were obtained from ATCC. Cells were maintained inthe undifferentiated state by culturing at low density in high glucoseDulbecco's Modified Eagle Media (DMEM) supplemented with 20% fetalbovine serum, 1% chicken embryo extract, 100 units/ml penicillin and 100μg/ml streptomycin. For some studies, myogenic differentiation wasinduced by culturing in DMEM supplemented with 2% horse serum andantibiotics. Immediately before injection, myoblasts were harvested withtrypsin, washed three times with serum free DMEM and directly injectedinto the ventricular myocardium of adult syngeneic C3Heb/FeJ mice(Jackson Laboratories) under open hieart surgery as described in (7).Cells (4-10×10⁴ were injected in a volume of 2-3 μl using a plasticsyringe fitted with a 30 gauge needle.

[0046] Histology.

[0047] Hearts were removed, cryoprotected, embedded and sectioned as inExample 1. H and E staining and [³H]-thymidine incorporation assays werealso conducted as in Example 1. For immunohistology, methanol fixedsections (−20° C., 10 min.) were reacted with the monoclonalanti-skeletal myosin heavy chain antibody (MY-32, Sigma Chemical Corp.)followed by rhodamine-conjugated sheep anti-mouse IgG F(ab′)₂ fragment(Boehringer Mannheim), and visualized by epifluorescence.

[0048] Electron Microscopy.

[0049] EM was performed as in Example 1.

[0050] Electrocardiogram Analyses.

[0051] ECG anlyses were performed as in Example 1.

[0052] Plasma Enzyme Assay.

[0053] PEA was performed as in Example 1.

[0054] B. Results

[0055] Several myoblast cell lines are known which, as exemplified byC2C12 cells, have the capacity to differentiate into myotubes in culture(13). C2C12 myoblasts were derived from cultured explants of injuredthigh muscle of C3H mice. When maintained in serum-rich media, themyoblasts proliferate rapidly and retain an undifferentiated phenotype.However, when cultured in serum-poor media myogenic differentiation isinduced. The C2C12 cells withdraw from the cell cycle and fuse, therebyforming multinucleated myotubes. Myogenic differentiation is alsoinduced, as evidenced by the appearance of numerous muscle-specific geneproducts. Thus, in this model proliferation and myogenic differentiationare mutually exclusive (14). Myoblast differentiation in vitro isthought to mimic satellite cell mediated myofiber regeneration in vivo.

[0056] Myoblasts were injected directly into the myocardium of syngeneicC3Heb/FeJ mice and the viability of the grafted material was assessed.One hundred percent (13/13) of the mice receiving intra-cardiac implantsof C2C12 myoblasts developed grafts in the heart. Viable grafts wereobserved as long as six months post-implantation (this was the last timepoint assayed). In all instances, the grafted material was notencapsulated. The differentiated status of the grafted C2C12 cells wasdetermined by immunohistological assay with an anti-myosin heavy chainantibody (MY-32). This antibody does not react with myoblasts nor withcardiac myosin heavy chain. Although differentiated C2C12 cells wereobserved in every heart receiving myoblast injections, the graftingefficiency of individual cells was not determined. As an additionalcontrol, hearts bearing AT-1 intra-cardiac grafts (see Example 1) wereexamined with the MY-32 antibody. No staining was observed, therebyruling out the possibility that the signal seen in the C2C12 grafts wasdue to skeletal myosin heavy chain induction in host cardiomyocytes.

[0057] Example 1 above demonstrates that AT-1 cardiomyocytes form stablegrafts in syngeneic myocardium. However, the observation that thesecells retained the capacity for proliferation in vivo raised thepossibility that sustained cell division might be required forsuccessful intra-cardiac grafting. The proliferative status of the C2C12grafts was therefore examined. Virtually no DNA synthesis (as assessedby tritiated thymidine incorporation) was observed, indicating that themajority of the grafted C2C12 cells had indeed withdrawn from the cellcycle. Examination of serial sections indicated that less than 0.1% ofthe cells in or near the grafts were synthesizing DNA. This result mostlikely reflects fibroblast proliferation during the remodeling process.As with the AT-1 grafts, immunohistological analyses of C2C12 graftsfailed to detect macrophage, inflammatory leukocyte or lymphocyteinfiltration at two months post-implantation, indicating the absence ofchronic graft rejection by the syngeneic hosts.

[0058] At the level of light microscopy, the C2C12 intra-cardiac graftsexhibited cellular heterogeneity with both H and E and MY-32immunofluorescence staining. Electron microscopic analyses were employedin an effort to further characterize the cellular make-up of the C2C12grafts. Toluidine-stained 1 μm sections were surveyed at 100 μmintervals to locate graft sites for EM analysis. Once localized, thinsections were prepared from the block. Cells with morphology typical ofskeletal myocytes were observed throughout the graft. Abundantmitochondria localized between well developed sarcomeres were readilydetected. Prominent Z bands and thick and thin filaments were observed.Occasionally, expanded t-tubules and ruffled cell membranes weredetected in the grafted myocytes. In addition to well developedmyocytes, a second less differentiated cell type was observed in C2C12grafts. Most notably, these cells exhibited a large nucleus to cytoplasmratio, with a prominent band of heterochromatin at the nuclearperiphery. Moderate amounts of centrally located heterochromatin werealso detected. Limited rough endoplasmic reticulum and few mitochondriawere observed in these cells. Similar ultrastructural characteristicshave been ascribed to satellite cells in vivo and in culture (15, 16).

[0059] Two studies were initiated to assess any deleterious effects ofC2C12 intra-cardiac grafts on host heart function. In the first study,surface electrocardiograms failed to detect any appreciable differencesbetween records from control and experimental mice. All animals examinedhad normal P-QRS coupling, and exhibited normal sinus rhythm with ananesthetized heart rate of approximately 400 beats per minute. Thesedata indicate that the intra-cardiac myoblast grafts did not induceovert cardiac arrhythmias. In the second study, plasma LDH levels weremonitored in graft-bearing animals. The presence of the cardiac LDHisoform in the circulation is a well established hallmark of myocardialinfarction. The cardiac-specific LDHI isoforms (isoforms 1, 2, and 3)were not observed in plasma prior to grafting. Immediately aftergrafting, an increase in the cardiac isoforms was observed in plasma,which most likely reflected damage to the host myocardium. A transientincrease in the plasma skeletal LDH isoform (isoform 5) was alsoobserved, presumably reflecting damage caused by the trans-thoracicincision. Plasma LDH profiles returned to normal by 7 dayspost-implantation.

EXAMPLE 3 Generation of Stable Fetal Cardiomyocyte Grafts

[0060] A. Methods

[0061] Cardiomyocyte Cell Culture and Myocardial Grafting Protocol.

[0062] Transgenic mice were generated which carry a fusion genecomprised of the α-cardiac myosin heavy chain (MHC) promoter and amodified β galactosidase (nLAC) reporter. To generate the MHC-nLACtransgenic mice, MHC-nLAC insert DNA (see FIG. 1) was purified byabsorption onto glass beads, dissolved at a concentration of 5 μg/ml,and microinjected into the nuclei of one cell inbred C3H3B/FeJ embryosaccording to established protocols (17). Polymerase Chain Reaction (PCR)analysis was employed to identify founder animals and to monitortransgene segregation. The sense strand primer5′-GGTGGGGGCTCTTCACCCCCAGACCTCTCC-3′ was localized to the MHC promoterEnd the antisense strand primer 5′-GCCAGGGTTTTCCCAGTCACGACGTTGT-3′ waslocalized to the nLAC reporter. PCR analyses were as described in (18).The MHC promoter consisted of 4.5 kb of 5′ flanking sequence and 1 kb ofthe gene encompassing exons 1 through 3 up to but not including theinitiation codon. The nLAC reporter was modified so as to carry both aeukaryotic translation initiation site and the SV40 nuclear localizationsignal (19). The mPl sequences carried an intron, as well astranscriptional termination and polyadenylation signals from the mouseprotamine 1 gene.

[0063] For preparations for examination of β galactosidase (βGAL)activity and DAPI epifluorescence, transgenic animals were heparinized(10,000 U/kg IP) prior to sacrifice by cervical dislocation. Hearts wereplaced in a beaker of gassed (95% O₂, 5% CO₂) KHB buffer (105 mM NaCl,20 noM NaHCO₃, 3.8 mM KCl, 1 mM KH₂PO₄, 1.2 mM MgSO₄, 0.01 mM CaCl₂, 1mM mannitol, 10 mM taurine, 10 mM dextrose, 5 mM Na-pyruvate). Heartswere then hung by the aorta and perfused with gassed KHB (0.5 ml/min at37° C.) containing 2.5 mM EGTA for five minutes, followed by 0.17%collagenase (Type I, Worthington Biochemical, Freehold N.J.) in KHB.Hearts were prefused until flaccid and the ventricles were minced withscissors and isolated cells obtained by triturating with a Pasteurpipette. After at least one hour of formalin fixation, suspensions werefiltered and smeared onto positively charged slides (Superfrost Plus,Fisher, Pittsburgh Pa.), and allowed to dry.

[0064] For isolation of single cells for injection, females with 15 dayembryos (onset of pregnancy determined by vaginal plugs) were sacrificedby cervical dislocation. Embryos were removed, decapitated, and heartswere harvested under PBS, and ventricles and atria were separated.Transgenic ventricles (identified by cardiac βGAL activity) weredigested in 0.1% collagenase (Worthington) in DPBS (Dulbecco's PhosphateBuffered Saline, Sigma) for 45 minutes, and were triturated with aPasteur pipette in PC-1 medium (Ventrex, Coons Rapids Minn.) with 10%FBS, resulting in a suspension of single cells.

[0065] Immediately after isolation, embryonic cardiomyocytes were washedthree times with DPBS and directly injected into the ventricularmyocardium of syngeneic mice (Jackson Laboratories) under open heartsurgery as in Example 1. 1-10×10⁴ cells were injected in a volume of 2-3μl using a plastic syringe fitted with a 30 gauge needle.

[0066] Histology.

[0067] For H and E, X-GAL, immunohistology and thymidine analyses,hearts were removed following cervical dislocation and cryoprotected in30% sucrose, embedded and sectioned at 10 μm with a cryomicrotome as inExample 1. H and E staining, monitoring for intra-cardiac graftrejection, and assay for [³H]-thymidine incorporation were alsoconducted as in Example 1. To assay βGAL activity, sections werehydrated in PBS, post-fixed in acetone:methanol (1:1) and then overlaidwith mixture containing 1 mg/ail X-GAL(5-bromo-4-chloro-3-indolyl-β-D-galactoside), 5 mM potassiumferricyanide, 5 mM potassium ferrocyanide and 2 mM magnesium chloride inPBS. Positive staining is indicated by the appearance of a bluechromophore. After treatment with primary antibody, signal wasvisualized by an avidin-biotin (ABC) kit (Vector Labs, BurlingameCalif.). The heart was processed as described above, and sections werepost-fixed in methanol:acetone (1:1), stained with H and E, and coatedwith a thin layer of photographic emulsion (Ilford L.4, Polysciences)diluted 1:1 with distilled water. Sections were exposed, developed,washed, fixed and washed as in Example 1. X-GAL staining of single cellpreparations was as described above. For visualization of nuclei insingle cell preparations, slides were stained with DAPI in PBS (0.28 μM,three min. at room temperature, Boehringer Mannheim), washed three timesin PBS, and wet-mounted in 2% propyl gallate dissolved in glycerol. Toobtain coronal heart sections, mice were sacrificed by cervicaldislocation, hearts were harvested and perfused on a Langendorffapparatus with 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4).After immersion fixation overnight in the same buffer, 200 μm coronalsections were made with a vibratome (Campden, London, United Kingdom).To localize the graft, sections were pooled and stained for βGALactivity with X-GAL as described above.

[0068] Electron Microscopy.

[0069] MHC-nLAC-embryonic grafts were localized in coronal heartsections as described above. After trimming, the tissue was post-fixedin 2% osmium tetroxide (Stevens Metallurgical Corp., New York N.Y.).Tissue was then dehydrated and embedded in Ladd LX-112 (Ladd ResearchIndustries). Grafted areas were further trimmed, thin sectioned, andstained with uranyl acetate and lead citrate. Specimens were viewed on aPhillips 400 transmission electron microscope as in Example 1.

[0070] Electrocardiogram Analyses.

[0071] ECG analyses were conducted as in Example 1.

[0072] B. Results

[0073] Transgenic mice generated as above carried a fusion genecomprised of the MHC promoter and a nLAC reporter. nLAC carries the SV40nuclear transport signal, which results in the accumulation of βgalactosidase activity in the nucleus of targeted cells. Four transgeniclineages were produced, and two (designated MHC-nLAC-2 and MHC-nLAC-4)were selected for further analyses. To ensure that the MHC-nLACtransgene would provide a suitable cell lineage marker, β galactosidase(βGAL) activity was assessed in transgenic cardiomyocytes. Single cellpreparations generated by retrograde collagenase perfusion were examinedsimultaneously for βGAL activity and DAPI epifluorescence. 99.0±0.45%(n=400) of the transgenic cardiomyocyte nuclei expressed βGAL, whereasno βGAL activity was detected in noncardiomyocytes. In addition, nonuclear βGAL activity was detected in nontransgenic controlcardiomyocytes.

[0074] Single cell suspensions were prepared by collagenase digestion ofhearts harvested from embryonic day 15 transgenic mice. Greater than 95%of the cardiomyocytes isolated by this technique were viable asevidenced by dye exclusion assay. Cardiomyocytes were delivered to leftventricular free wall of syngeneic nontransgenic animals. Graftedcardiomyocytes were readily and unambiguously identified by virtue ofthe nuclear βGAL activity encoded by the MHC-nLAC transgene. Graftedcardiomyocytes were frequently observed at sites distal to the point ofdelivery; it presently is not clear if this distribution of graftedcells reflects cardiomyocyte migration or passive diffusion alongdissection planes produced by the injection process. Approximately 50%(7/13) of the animals receiving intra-cardiac injections of embryoniccardiomyocytes developed grafts. This frequency of successful graftformation is likely to increase as cell preparation and implantationprotocols are optimized.

[0075] Light microscopic analyses of H and E stained sections processedfor βGAL activity indicated that grafted cardiomyocytes (blue nuclei)were juxtaposed directly with host cardiomyocytes (purple nuclei).Additional H and E analyses failed to detect significant graftencapsulation. The observed proximity of graft and host cardiomyocytesand absence of encapsulation are prerequisites for successful couplingbetween the two cell types.

[0076] Consecutive sections of a 19 day old intra-cardiac graft wereprocessed for H and E, βGAL activity, and macrophage and leukocyteimmunoreactivity. No evidence for graft rejection was observed, despitethe fact that the animals were not immune suppressed. As a positivecontrol for the immunohistology, grafts of incompatible MHC haplotypewere produced; graft rejection was clearly evident in these hearts.Tritiated thymidine uptake analyses indicated that only 0.6% (n=156) ofthe βGAL-positive nuclei were synthesizing DNA, althoughnoncardiomyocyte DNA synthesis was apparent. Since the embryonic day 15donor cells were still mitotically active when grafted (labeling indexof ca. 29%), the exceedingly low level of DNA synthesis observed in βGALpositive cells at 19 days post-grafting suggested that the MHC-nLACembryonic cardiomyocytes had undergone terminal differentiation.

[0077] The juxtaposition of graft and host cardiomyocytes observed bylight microscopic analyses prompted a determination whether directintercellular coupling could be detected between the two cell types. TheX-GAL reaction product is an electron-dense precipitate which can bedetected by transmission electron microscopy (TEM, see 19). Vibratomesections from glutaraldehyde perfusion-fixed hearts were stained forβGAL activity, and grafted regions thus identified were trimmed andembedded for TEM. βGAL positive nuclei were readily observed by lightmicroscopic analysis of 1 μm sections. The X-GAL reaction product had aperinuclear appearance due to a slight degree of nuclear leaching whichoccurred during the embedding process. The sable groups ofcardiomyocytes were identified by TEM analysis of a consecutive thinsection. Host cardiomyocytes, which were not readily identified in thelight micrographs due to the absence of perinuclear βGAL activity, wereobserved by electron microscopy to be juxtaposed with the grafted cells.Numerous junctional complexes were present between the host and graftcardiomyocytes, indicating a high degree of intercellular coupling. Manyexamples of intercellular coupling between host and graft cardiomyocyteswere observed throughout the grafted regions. Importantly, intercellularconnections could be traced from βGAL positive cardiomyocytes throughnumerous host cells, thus demonstrating that grafted cardiomyocytescould be participating in a functional syncytium.

[0078] In addition to documenting the presence of abundant intercellularcoupling between grafted and host cardiomyocytes, the TEM analysesrevealed that the grafted cardiomyocytes were highly differentiated.Normal characteristics of adult cardiomyocytes were observed includingmyofibrillae forming complete sarcomeres, numerous junctional complexesbetween cells and abundant mitochondria. Indeed, aside from the presenceof the X-GAL reaction product, grafted cardiomyocytes wereindistinguishable from host cells. Further, binucleated, βGAL positivecells could be detected in the intra-cardiac grafts. Becausebinucleation is a characteristic of adult rodent cardiomyocytes, thisobservation further supports that the grafted cardiomyocytes haveundergone terminal differentiation.

[0079] Surface ECG recordings were employed to determine if the presenceof coupled embryonic cardiomyocyte grafts negatively influenced hostheart automaticity. ECG traces from graft-bearing animals wereindistinguishable from sham operated controls, and exhibited P and QRScomplexes typical for mice. There was no evidence for cardiac arrhythmiain graft-bearing animals, despite the presence of a high degree ofintercellular coupling between grafted and host cardiomyocytes.

EXAMPLE 4 Preparation of Substantially Pure Cardiomyocyte Culture

[0080] Embryonic stem cells were genetically modified in a mannerenabling the production of a substantially homogeneous population ofnon-immortalized cardiomyocytes. The parental ES cell line (D3) wascotransfected with a pGK-HYG (hygromycin) plasmid and a plasmidcontaining a MHC-neo^(r) gene. The pGK-HYG plasmid provides selectionfor transfected ES cells, while the mMHC-neo^(r) gene facilitates asecond round of selection on differentiated cells: incubation in thepresence of G418 eliminates non-cardiomyocytes (that is, cells in whichthe MHC promoter is not active).

[0081] Stably tranfected ES cells were selected by growth in thepresence of hygromycin. The plasmids were linearized and introduced intothe stem cells via electroporation at 1180 μFarad, 220 volts. Thetransfected cells were maintained in DMEM supplemented with 10%preselected FBS, 0.1 mM β-mercaptoethanol, nonessential amino acids,PenStrep and LIF, and transformants selected by the addition ofhygromycin into the medium. Co-transfectants were then identified by PCRanalysis specific for both transgenes. The transfections produced a cellline, designated 9A, which carries both transgenes.

[0082] Cardiogenesis was induced in 9A ES cells by plating 2×10⁶ cellsonto uncoated 100 mm bacterial petri dishes in the absence of LIF. After8 days in culture, numerous patches of cells exhibiting spontaneouscontractile activity (i.e. cardiomyocytes) were observed. At this point,G418 was added to the media, and the cells incubated for an additional 9days. During this treatment it was apparent that many of thenon-cardiomyocytes were being killed by the G418. Importantly, the G418had no discernible effects on the cardiomyocytes, which retained theirspontaneous beating activity throughout the course of the experiment.

[0083] After a total of 9 days of G418 selection, the surviving cellswere dissociated with collagenase and trypsin, and then replated ontofibronectin coated microscope slides. The cells were cultured anadditional 24 hours to allow them to recover from passage, and thenfixed for immunocytologic analysis. Cells were reacted with MF20, amonoclonal antibody which recognizes sarcomeric myosin. Cells were thenindividually counted for the presence or absence of sarcomeric myosin, amarker for cardiomyocytes. The results were as follows: Total number ofcells counted:  794 Number of MF20+ cells:  791 Number of MF20− cells:  3* Percent cardiomyocytes: 99.6%

[0084] It was thus demonstrated that the expression of drug (neomycin)resistance under a cardiac-specific promoter enables the selection of anessentially pure population of ES derived cardiomyocytes in culture.Such populations can be used to form myocardial grafts using proceduresas discussed in the Examples above.

EXAMPLE 5 Delivery of Protein via Graft

[0085] A. Methods

[0086] C2C12 Cell Culture and Transfection.

[0087] C2C12 myoblasts (ATCC) were maintained in the undifferentiatedstate by culturing at low density in high glucose Dulbecco's ModifiedEagle Media (DMEM)¹ supplemented with 20% fetal bovine serum, 1% chickenembryo extract, 100 units/ml penicillin and 100 μg/ml streptomycin. Forsome studies, myogenic differentiation was induced by culturing in DMEMsupplemented with 2% horse serum and antibiotics.

[0088] A fusion gene comprised of the metallothionein (MT) promoterdriving a modified Transforming Growth Factor-Beta 1 (TGF-β1) cDNA wasobtained from Samuel and colleagues (20). Transcriptional activity ofthe metallothionein promoter can be regulated by modulating the heavymetal content of cell culture media. The TGF-β1 cDNA carriedsite-directed mutations which resulted in the conversion of Cys²²³ andCys²²⁵ to serines. This modification (described further in (21)) resultsin the elaboration of a TGF-β1 molecule which is unable to form dimers,and consequently is not subject to normal post translational regulation.Cells expressing the modified cDNA constitutively secrete processed,active TGF-β1 (20). The MT-TGF fusion gene was introduced into C2C12myoblasts by calcium phosphate transfection; stable transfectarits wereselected by virtue of co-transfection with an SV40-neo^(r) transgene.Four independent clones were isolated, and presence of the transgene wasconfirmed by Southern blot analysis. The relative levels of TGF-β1expression in the different clonal cell lines was initially assessed byNorthern blot analysis, and one line, designated C2(280), was utilizedfor subsequent experiments.

[0089] Myocardial Grafting Protocol.

[0090] The grafting protocol was as described in Example 1. Fourteendays post-surgery, graft bearing animals were given heavy metal (25 mMZnSO₄ in drinking water). Zinc treatment was continued until thetermination of the experiment (1-4 weeks).

[0091] Histology.

[0092] For paraffin sections, hearts were fixed in 10% neutral bufferedformalin, dehydrated through graded alcohols, and infiltrated withparaffin. Tissue blocks were then sectioned at 6 μm. H and E stainingwas performed directly after sectioning according to manufacturer'sspecifications (Sigma Diagnostics).

[0093] For [³H]-thymidine incorporation, mice were given a bolus andsacrificed as in Example 1. The heart was removed and processed forparaffin embedding as described above. Autoradiography was likewiseconducted as iii Example 1.

[0094] B. Results

[0095] Expression of recombinant TGF-β1 in response to heavy metalinduction was examined in C2(280) myoblasts and myotubes. Transgenetranscripts (1.8 kb) were readily distinguished from those originatingfrom the endogenous TGF-β1 gene (2.5 kb) by Northern blot analysis.Addition of heavy metal to the culture media resulted in a markedincrease of recombinant TGF-β1 transcripts in C2(280) myoblasts andmyotubes. As indicated above, modified TGF-β1 expressed by C2(280) cellsshould have constitutive biological activity. To directly test this,conditioned media from C2(280) myoblasts and myotubes was examined bygrowth inhibition assay.

[0096] C2(280) myoblasts were used to produce intra-cardiac grafts insyngeneic C3Heb/FeJ mice. The presence of grafts was readily detected inH and E stained sections. 100% (n>50) of the animals receivingintra-cardiac injections of C2(280) cells went on to develop grafts.Interestingly, H and E analysis suggested that the C2(280) grafts weresomewhat less differentiated as compared to those produced withunmodified C2C12 cells. This result was confirmed by immunohistologicanalysis with a monoclonal antibody which recognizes skeletal myosinheavy chain.

[0097] C2(280) graft transgene expression was assessed byimmunohistology with an anti-TGF-β1 antibody; TGF-β1 expression wasreadily detected in C2(280) grafts. As a negative control, TGF-β1expression was assessed iii grafts produced by C2C12 myoblasts. Asexpected, the relative levels of TGF-β1 expression were markedly reducedin C2C12 grafts as compared to C2(280) grafts.

[0098] TGF-β1 is a well known angiogenic factor. ³H-thymidineincorporation analyses in vascular endothelial cells was thereforeassessed to determine if an enhanced angiogenic response occurred ingrafts expressing the MT-TGF transgene. DNA synthesis in vascularendothelial cells was readily apparent in C2(280) grafts underadministration of a single bolus injection of ³H-thymidine (H-THY). Incontrast, vascular endothelial DNA synthesis was markedly reduced innon-transfected C2C12 grafts (Table 1). To rule out the possibility thatthe angiogenic responses was due solely to graft mass, ³H-thymidineincorporation was compared between similar size and aged C2C12 andC2(280) grafts (Table 1). A marked increase in the number of vascularendothelial cells synthesizing DNA was apparent in all of the analyses.Finally, the thymidine incorporation assay also revealed that apercentage of the grafted myoblasts continued to proliferate. Thisobservation is consistent with the known inhibitory effect of TGF-β1 onmyodifferentiation, and most likely accounts for the undifferentiatedappearance of the C2(280) grafts. TABLE 1 TGF-β1 Delivery and VascularEndothelial DNA Synthesis Time Post Zn C2C12 C2(280) Induction TGF-β1(−) TGF-β1 (+) 1 Week Total # ³H-THY +  0  8 Endothelial Cells Total #of Vessel 35 17 Sections Counted # Synthetic Cells/ 0.0 ± 0.00 0.46 ±0.036 Vessel Section 2 Weeks Total # ³H-THY +  1  7 Endothelial CellsTotal # of Vessel 18 21 Sections Counted # Synthetic Cells/ 0.06 ± 0.0560.34 ± 0.052 Vessel Section

[0099] While the invention has been illustrated and described in detailin the foregoing description, the same is to be considered asillustrative and not restrictive in character, it being understood thatonly the preferred embodiments have been described and that allmodifications that come within the spirit of the invention are desiredto be protected.

REFERENCES

[0100] The following references, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are specifically incorporated herein by reference.

[0101] 1. Tompson, L. Fetal transplants show promise. Science 257:868-870, 1992.

[0102] 2. Gussoni, E., Pavlath, G. K., Lanctot, A. M., Sharma, K. R.,Miller, R. G., Steinman, L. and Blau, H. M. Normal dystrophintranscripts detected in Duchenne muscular dystrophy patients aftermyoblast transplantation. Nature 356: 435-438, 1992.

[0103] 3. Steinhelper, M. E., Lanson, N., Dresdner, K., Delcarpio, J.B., Wit, A., Claycomb, W. C. and Field, L. J. Proliferation in vivo andin culture of differentiated adult atrial cardiomyocytes from transgenicnice. American Journal of Physiology 259 (Heart and CirculatoryPhysiology 28): H1826-H1834, 1990.

[0104] 4. Janse, M. J., Cinca, J., Morena, H., Fiolet, J. W., Kleber, A.G., deVries, G. P., Becker, A. E. and Durrer, D. The border zone inmyocardial ischemia. An electrophysiological, metabolic andhistochemical correlation in the pig heart. Circulation Research44:576-588, 1979.

[0105] 5. Spear, J. F., Michelson, E. L., and More, E. N. Cellularelectrophysiologic characteristics of chronically infarcted myocardiumin dogs susceptible to sustained ventricular tachyarrhythmias. Journalof the American College of Cardiology 4:1099-1110, 1983.

[0106] 6. Delcarpio, J. B., Lanson, N. A. Jr., Field, L. J. andClaycomb, W. C. Morphological characterization of cardiomyocytesisolated from a transplantable cardiac tumor derived from transgenicmouse atria (AT-1 cells). Circulation Research 69:1591-1600, 1991.

[0107] 7. Rockman, H. A., Ross, R. A., Harris, A. N., Knowlton, K. U.,Steinhelper, M. E., Field, L. J., Ross, J. Jr. and Chien, K. R.Segregation of atrial-specific and inducible expression of an ANFtransgene in an in vivo murine model of cardiac hypertrophy. Proc. Natl.Acad. Sci. USA 88:8277-8281, 1991.

[0108] 8. Bullock, G. R. and Petrusz, P., in Techniques inImmunocytochemistry, Vol. II, Academic Press, New York, 1983.

[0109] 9. Field, L. J. Atrial natriuretic factor-SV40 T antigentransgenes produce tumors and cardiac arrhythmias in mice. Science239:1029-1033, 1988.

[0110] 10. Katz, E., Steinhelper, M. E., Daud, A. Delcarpio, J. B.,Claycomb, W. C. and Field, L. J. Ventricular cardiomyocyte proliferationin transgenic mice expressing α-Cardiac Myosin Heavy Chain-SV40 Tantigen fusion genes. American Journal of Physiology 262 (heart andCirculatory Physiology 31):H1867-1876, 1992.

[0111] 11. Steinhelper, M. E. and Field, L. J. “SV40 large T-Antigeninduces myocardiocyte proliferation in transgenic mice”, in Thedevelopment and regenerative potential of cardiac muscle, JohnOberpriller and Jean Oberpriller, eds. Harwood Academic press, 1990.

[0112] 12. Steinhelper, M. E. and Field, L. J. Cardiac Tumors anddysrhythmias in transgenic mice. Toxicologic Pathology 18: 464-469,1990.

[0113] 13. Yaffe, D., and O. Saxel. Serial passaging and differentiationof myogenic cells isolated from dystrophic mouse muscle. Nature270:725-727, 1977.

[0114] 14. Nadal-Ginard, B. Commitment, fusion, and biochemicaldifferentiation of a myogenic cell line in the absence of DNA synthesis.Cell 15:885-864, 1978.

[0115] 15. Bruni, C. Mitotic activity of muscle satellite cells duringthe early stages of rhabdomyosarcomas induction with nickel subsulfide.In Muscle Regeneration, A. Mauro, editor. Raven Press, New York, N.Y.,1979.

[0116] 16. Rubin, L. L., C. E. Keller and S. M. Schuetze. Satellitecells in isolated adult muscle fibers in tissue culture. In MuscleRegeneration, A. Mauro, editor. Raven Press, New York, N.Y., 1979.

[0117] 17. B. Hogan, F. Costantini and E. Lacy, in Manipulating theMouse Embryo—A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986.

[0118] 18. M. E. Steinhelper, K. L. Cochrane and L. J. Field.Hypotension in transgenic mice expressing atrial natriuretic factorfusion genes. Hypertension 16:301-307, 1990.

[0119] 19. A. D. Loewy, P. C. Bridgman and T. C. Mettenleiter. BrainResearch 555:346, 1991.

[0120] 20. S. K. Samuel, et al. Autocrine induction of tumor proteaseproduction and invasion by a metallothionein-regulated TGF-beta 1(Ser223,225). Embo Journal [JC:emb] 11(4):1599-1605, 1992.

[0121] 21. A. M. Brunner, et al. Site-directed mutagenesis of cysteineresidues in the pro region of the transforming growth factor beta 1precursor. Expression and characterization of mutant proteins. Journalof Biological Chemistry 264(23):13660-13664, 1989.

[0122] 22. E. J. Robertson. Embryo-derived stem cell lines, inTeratocarcinomas and embryonic stem cells: a practical approach, E. J.Robertson, editor. IRL Press, Washington DC, 1987.

What is claimed is:
 1. A myocardial graft in an animal, comprising: astable graft of skeletal myoblasts or cardiomyocytes incorporated inmyocardial tissue of said animal.
 2. The myocardial graft of claim 1wherein said stable graft comprises cardiomyocytes.
 3. The myocardialgraft of claim 1 wherein said stable graft comprises skeletal myoblasts.4. The myocardial graft of claim 1 which is non-tumorogenic.
 5. Themyocardial graft of claim 1 wherein said stable graft deliversrecombinant molecules to the myocardial tissue.
 6. The myocardial graftof claim 5 which is non-tumorogenic.
 7. A method for forming a stablemyocardial graft. in an animal, comprising: introducing skeletalmyoblasts or cardiomyocytes in myocardial tissue of the animal so as toform a stable myocardial graft.
 8. The method of claim 7 wherein saidintroducing comprises injecting the skeletal myoblasts or cardiomyocytesinto myocardial tissue of the animal.
 9. The method of claim 7 in whichskeletal myoblasts are introduced into the myocardial tissue.
 10. Themethod of claim 7 in which cardiomyocytes are introduced into themyocardial tissue.
 11. The method of claim 7 wherein the myocardialgraft is non-tumorogenic.
 12. The method of claim 11 wherein themyocardial graft delivers recombinant molecules to the myocardialtissue.
 13. A method for delivering recombinant molecules to myocardialtissue of an animal, comprising: establishing a stable graft of skeletalmyoblasts or cardiomyocytes incorporated in myocardial tissue of theanimal, wherein the myoblasts or cardiomyocytes deliver recombinantmolecules to the myocardial tissue.
 14. The method of claim 13 whereinthe stable graft comprises skeletal myoblasts.
 15. The method of claim13 wherein the stable graft comprises cardiomyocytes.
 16. The method ofclaim 13 wherein the graft is non-tumorogenic.
 17. A cellularcomposition comprising a substantially homogeneous population ofnon-immortalized cardiomyocytes.
 18. A method of obtaining asubstantially homogeneous population of cells, comprising: transfectingembryonic stem cells to introduce a marker gene enabling selection ofone cell lineage from other cell lineages resulting from thedifferentiation of the stem cells; causing the stem cells todifferentiate; and selecting said one cell lineage based on said markergene.
 19. The method of claim 18 , comprising: transfecting the stemcells to introduce (i) a first marker gene enabling selection oftransfected stem cells from non-transfected stem cells and (ii) a secondmarker gene enabling selection of said one cell lineage from said othercell lineages; selecting transfected stem cells based on the firstmarker gene; causing the selected'stem cells to differentiate; andselecting said one cell lineage based on said marker gene.
 20. Themethod of claim 19 wherein said one cell lineage is cardiomyocytes. 21.A non-human animal having a stable graft of skeletal myoblasts orcardiomyocytes incorporated in myocardial tissue of the animal.
 22. Theanimal of claim 21 which is a mammal.
 23. The animal of claim 22 whereinthe graft is non-tumorogenic.
 24. The animal of claim 23 wherein thegraft includes cardiomyocytes.
 25. The animal of claim 23 wherein thegraft includes skeletal myoblasts.